Plants have distinctive embryologic characteristics different from those of other eukaryotes. Since plant cells do not migrate, cell division, extension, and program cell death are thought to determine morphogenesis. Cells proliferate in meristematic tissues existing at both poles of a shoot apex and a root apex. The proliferated cells differentiate and pile up to develop into a plant. The size of a plant is determined by the number and size of constituent cells for forming the plant. Plants control cell growth (cell proliferation) by regulating the cell cycle so that the sizes of plants are adapted to changed environmental conditions. The regulation of the cell cycle is important for differentiation of plants. For example, pericyclic cells of roots remain in a specific period (G2 phase) of the cell cycle, and differentiation of lateral roots is determined by whether or not the cells start to divide. The number of the hypocotyl cells is regulated in plants, but the cell cycle changes in the dark and the cell size changes by endoreduplication.
A method for controlling cell division of plants, in particular, a method for regulating the cell cycle is thought to be important as a new method of plant breeding which collectively treats matters such as growth, shapes, and stress response.
A cell is divided into two daughter cells through a series of processes called a cell cycle that consists of four phases; i.e. gap 1 phase (G1 phase), DNA synthesis phase (S phase), gap 2 phase (G2 phase), and mitosis phase (M phase). Mechanisms relating to the regulation of S and M phases of the cell cycle have been noticed and studied. In the two phases, M phase, called mitotic division phase, is a phase for equally distributing the chromosomes duplicated in the S phase into daughter cells. For entry into the M phase, cyclins (one representative is Cyclin B) bind to cyclin-dependent kinase (CDK) to form an activated complex for enhancing aggregation of chromosomes and disruption of nuclear membranes. The M phase terminates after a process called cytokinesis (cytoplasmic division) for dividing cytoplasm after the distribution of the chromosomes into two. In plant cells, phragmoplast which is a structure specific to plant is formed and the cytoplasmic division progresses. The formation of phragmoplasts is regulated by kinesin-like proteins; i.e. NACK1 and NACK2.
Cyclin B, NACK1, and NACK2 having important functions in the process from the entry into the M phase till the termination of the M phase in plant cells show gene expression patterns specific to G2/M phases. It is reported that a specific control sequence called M-specific activator (MSA) existing in a promoter region controls the phase-specific expression of these genes (Non-patent Document 1). In addition to a plant-specific CDK, CDKB, and genes having high similarities to cyclin-specific E2 enzymes among E2 enzymes relating to proteolysis, a variety of functionally unknown genes have been reported to have M phase-specific expression patterns. Many of these genes are analyzed to include MSA sequences in promoter regions. Therefore, it is thought that mechanisms for regulating G2/M phase-specific gene expression by MSA sequences are universally conserved in plants.
NtmybA1, NtmybA2, and NtmybB (hereinafter collectively referred to as “Ntmyb”) have been identified from tobacco as MSA binding factors. The amino acid sequences of Ntmyb proteins characteristically have high similarities to the myb DNA binding region having a sequence composed of imperfect three repeats existing in animal c-myb and others (such proteins containing this DNA binding region are hereinafter referred to as “3Rmyb”). Many plants have genes carrying myb-like DNA binding regions, but most of them are constituted of two myb region repeats or non-repeated type myb regions. For example, Arabidopsis thaliana of which genome sequencing was completed has more than one hundred myb-like DNA binding region-containing genes, but only five genes contain the aforementioned myb-like DNA binding region composed of three imperfect myb repeats (3Rmyb). Thus, such genes are known as specific members among superfamilies constituting the plant myb-like protein group (Non-patent Document 2).
Transcription control experiments with reporter genes were conducted for investigations on Ntmyb functions using transient expression systems in plant cells. It was reported that NtmybA1 and NtmybA2 activated transcription of the Madagascar Periwinkle (Catharanthus roseus) cyclin B (CYM) promoter and the NACK 1 promoter, and vice versa NtmybB suppressed these transcription, thereby indicating that Ntmyb were capable of binding to MSA, and acted as transcription-controlling factors for genes exhibiting G2/M phase-specific expression (Non-patent Document 3). However, these reports are merely based on the results of reporter gene transcription activated by Ntmyb transiently expressed at one point in the cell cycle regulated by cyclic expression of numerous genes. Thus, there have been no reports disclosing functions of Ntmyb in the cell cycle and cell division yet.
There have been report examples disclosing that the growth and development of plants was modified by transformation with G2/M phase-specific expression genes. For example, the elongation of roots was enhanced in transformed plants which ectopically expressed cyclin B (Non-patent Document 4); and cytoplasmic division was incomplete to shorten plant height in plants having suppressed expression of NACK1 essential for the termination of the M phase or in transformed plants having dominant negative NACK1 constructs (Non-patent Document 5). However, these examples are the approaches for controlling the cell growth by utilizing individual genes relating to the progress of M phase. Therefore, there have been no reports disclosing transformed plants in which expression of G2/M phase-specific genes including functionally unknown genes, regulated by MSA sequences, is collectively regulated.
Ntmyb contains a myb DNA-binding region having high homology to c-myb. It is thought that the transcriptional function of c-myb is inactive when the EVES motif existing in said protein is binding to the myb DNA-binding domain, and is activated when phosphorylation of the EVES motif with a protein kinase leads to a change in the protein conformation, thereby allowing the binding of coactivator P100 to the myb DNA-binding region (Non-patent Document 6). Since regions other than the myb DNA-binding regions are observed to be nonsimilar between Ntmyb and c-myb, and regulating sequences such as the EVES motif are not conserved, the mechanism for controlling Ntmyb ability to activate transcription is thought to be different from that for c-myb protein. There have been no reports disclosing the presence of a region for controlling the transcription-activating ability of Ntmyb.
DNAs encoding full-length 3Rmyb have been reported in only tobacco and Arabidopsis thaliana which are dicotyledonous plants, but there have been no reports on full-length 3Rmyb from monocotyledonous plants. This means that there are no reports revealing whether or not mechanisms for regulating G2/M phase-specific gene expression, mediated by MSA sequence and 3Rmyb, are conserved in monocotyledonous plants and dicotyledonous plants.
Most of animals are diploid, but various ploidy levels are broadly known in plants. Triticum is hexaploid, and Asterales is decaploid. A large number of these polyploidy plants generally have characters or properties useful for agriculture, and creation of ploidy plants is used as a tool for breeding. Colchicine treatment has been broadly used as a technology for generating polyploids. Namely, colchicine treatment is performed to plant seeds, embryo plants, or tissue culture cells of organs, and then plants are selected from regenerated plants. Colchicine inhibits spindle formation of ploidy cells after DNA duplication, and ploidy cells are generated by skipping the mitosis phase. However, investigations of organs to be treated with colchicine and of timing for drug-treatment are necessary; thus, it is not readily performed in all plants.
[Non-patent Document 1] Ito et al., Plant Cell, 10: 331 (1998)
[Non-patent Document 2] Stracke et al., Curr. Opin. Plant Biol. 4: 447 (2001)
[Non-patent Document 3] Ito et al., Plant Cell, 13: 1891 (2001)
[Non-patent Document 4] Doerner et al., Nature, 380: 520 (1996)
[Non-patent Document 5] Nishihama et al., Cell, 109: 87 (2002)
[Non-patent Document 6] Dash et al., Genes Dev., 10: 1858 (1996)